| Hepatic fibrosis is a pathological process with excessive precipitation of extra cellular matrix（ECM）caused by various pathogenic factors.Preventing or slowing the development of hepatic fibrosis is prerequisite to the reduction of occurrence of liver cirrhosis.Currently,steroids,flavonoids and other chemical drugs are primarily applied for hepatic fibrosis treatment.Although of limited efficacy,they usually have the problems of severe side effects and drug resistance.Thus,there is an urgency to fill the clinical need using an effective treatment modality with mild or little side effects,as well as little drug resistance.Omega-3 polyunsaturated fatty acids（ω-3 PUFAs）are derived from plants and ocean fish,which are mainly composed ofα-linolenic acid（ALA）,eicosapentaenoic acid（EPA）,and docosahexaenoic acid（DHA）.Previous studies have confirmed thatω-3PUFAs can impose a variety of regulatory effects on inflammation.In recent years,the research ofω-3 PUFAs has been focused on various human chronic diseases.As an imporatant cellular growth regulator,the mammalian target of rapamycin（mTOR）signaling pathway regulates the activation and proliferation of hepatic stellate cells（HSCs）and the expression of collagen fibers.It is one of the signal pathways closely related to the occurrence and development of hepatic fibrosis.The protective effect ofω-3 PUFAs dietary intervention on hepatic fibrosis and its involvement on mTOR signaling pathway is still unclear,so further research is needed.The purpose of this study is to investigate the protective effect ofω-3 PUFAs dietary intervention on hepatic fibrosis and its involvement on mTOR signaling pathway,and to provide a experimental and mechanistic basis for potential new anti-fibrosis therapy.Objective:To investigate the protective effect ofω-3 PUFAs dietary intervention on hepatic fibrosis and the involvement of mTOR signaling pathway,which can provide a theoretical basis for its potential molecular mechanisms.Methods:Twenty male C57BL/6 mice were randomly divided into the control group,the 4-week model group,the 8-week model group and the treatment group,the 4-week model group was set up to determine the degree of hepatic fibrosis at the beginning ofω-3 PUFAs diet intervention.The mice in the model group and the treatment group were intraperitoneally injected with 20%CCl4 corn oil solution twice a week at a dose of 5ml·kg-1,whereas the mice in the control group were injected with the corn oil.Except for the 4-week model group,the other three groups were injected with corn oil or 20%CCl4corn oil for 8 weeks.Specifically,the treatment group were switched to an EPA/DHA-enriched diet since the fourth week of modeling,whereas the control group and the 8-week model group were given saline.（1）We recorded the weight during the modeling and the coat color,the grossing of the liver,and the liver to body weight ratios of the mice after the modeling to determine the degree of hepatic fibrosis model.We then used Masson three-color staining to evaluate the hepatic fibrosis;（2）HE staining were applied to detect the pathological changes of the liver tissues of mice in each group;（3）IHC assays were used to detect the expression of anti-apoptotic proteins Bcl-2,Bcl-x L and cell proliferation protein PCNA in the liver tissues of mice in each group;（4）IHC and WB assays were used to detect the expression of HSCs activation marker proteinα-SMA in the liver tissues of mice in each group;（5）Sirius red staining and WB assays were used to detect the content of collagen fibers and the expression of Collagen1A1 and Fibronectin in the liver tissues of mice in each group;（6）WB analysis was used to examine the total protein expression and the phosphorylation level of mTOR,S6 protein in the liver tissues of mice in each group.Results:（1）Compared with the control group,the weight,coat color,the gross shape of the liver,the liver to body weight ratios and the Masson three-color staining showed that the liver tissues in the 4-week model group developed fibrosis,indicating the successful modeling of hepatic fibrosis after 4 weeks;（2）Compared with the 8-week model group,the liver tissue damage of the treatment group was significantly improved,and the number of hepatocyte necrosis and inflammatory cells were significantly reduced,indicating thatω-3 PUFAs dietary intervention significantly improved fibrotic liver tissue damage;（3）Compared with the 8-week model group,the protein expression of Bcl-2,Bcl-x L and PCNA in the treatment group was significantly increased,indicating thatω-3PUFAs dietary intervention significantly inhibit hepatocyte apoptosis and enhance hepatocyte proliferation;（4）Compared with the 8-week model group,the treatment group had a significant reduction in the expression ofα-SMA,indicating that theω-3 PUFAs dietary intervention has an inhibitory effect on the activation of HSCs;（5）Compared with the 8-week model group,the treatment group had a significant reduction in the expression of collagen fibers,Collagen1A1 and Fibronectin,indicating that theω-3 PUFAs dietary intervention has a significant anti-fibrotic effect;（6）In addition,the phosphorylation levels of mTOR and S6 in the liver tissues of the treatment group decreased significantly,indicating that theω-3 PUFAs dietary intervention has an inhibitory effect on the mTOR signaling pathway in the liver tissues,and the mTOR signaling pathway is one of the signaling pathways mediating the protective effect.Conclusion:ω-3 PUFAs dietary intervention has a protective effect against hepatic fibrosis,mTOR signaling pathway is one of its potential molecular mechanisms. |
